The increasing prevalence of overweight and obese women of childbearing age is a growing public health concern, which can result in harmful, persistent effects in offspring, including pre-disposition to obesity and diabetes. Mechanisms linking maternal obesity and over nutrition to obesity and diabetes of offspring remain poorly defined. Skeletal muscle is the main periphery tissue responsible for glucose and fatty acid utilization. Since there is no increase in muscle fiber number after birth, fetal stage is crucial for skeletal muscle development. Fetal muscle up to late gestation contains a large number of mesenchymal stem cells (pluripotent progenitor cells). The proliferation and lineage commitment of pluripotent cells directly affect the number and size of muscle fibers developed. The commitment of pluripotent cells to adipogenesis instead of myogenesis will increase the number of intramuscular adipocytes, an event associated with insulin resistance in skeletal muscle. In our preliminary studies, we observed that maternal obesity and over-nutrition down-regulated AMP-activated protein kinase (AMPK) activity and insulin signaling, and promoted adipogenesis which should be associated with altered proliferation and differentiation of pluripotent progenitor cells. AMPK may play an important role in the control of proliferation and differentiation of pluripotent cells in fetal muscle in vivo. To confirm it, a good in vivo experimental model is needed. To establish an experimental model which can track the proliferation and differentiation of pluripotent cells in fetal muscle in vivo and to use this model to test the role of AMPK in fetal muscle development. Mesenchymal stem cells injected into fetal muscle will proliferate and differentiate into myogenic, adipogenic and other cells in fetal muscle. Mesenchymal stem cell C3H10T1/2 will be transfected with a pDsRed vector carrying AMPK constant active construct and a pAcGFP vector carrying dominant negative AMPK constructs respectively. pDsRed and pAcGFP vectors are mammalian expression vectors and express red and green fluorescent proteins respectively. Cells stably expressing red or green fluorescent proteins will be cloned and then injected into the skeletal muscle of fetuses. Pregnant sheep carrying these fetuses will be allowed to lamb which will then be euthanized for muscle collection. Muscle will be sectioned and the existence of fluorescent cells in muscle fibers, intramuscular adipocytes and connective tissues in skeletal muscle will be measured. The use of cells carrying two different fluorescent proteins will allow a direct comparison for the destination of cells with different AMPK activities. This model will be crucial for understanding the role of AMPK in the proliferation and differentiation of pluripotent cells in vivo. This model will also be useful for studying intracellular signaling pathways which affect differentiation of pluripotent cells in fetal muscle. Such studies will have enormous biomedical implications and will greatly facilitate studies regarding fetal developmental programming. [unreadable] [unreadable] [unreadable]